ZP3, a glycoprotein constituent of the, egg's zona pellucida, regulates the initiation of sperm acrosomal exocytosis during mammalian fertilization. The long range goal of this laboratory is to understand the mechanism of ZP3 signal transduction. The current phase of this research focuses on the regulation of sperm ion channels by ZP3 as well as on the relationship between ion channel activation and the elevations of internal Ca,+ that are required for exocytosis. These studies utilize digital image processing-enhanced fluorescence microscopy, fluorometry, and patch electrode-voltage clamping in order to explore ion channel regulation in living sperm. There are four specific aims. 1) The ionic basis of sperm membrane potential will be established. These studies will involve selective ion depletion and antagonist treatments to determine the principal ion permeabilities that establish membrane potential in resting cells. 2) The effects of ZP3 on sperm membrane potential will be determined, by identifying ion channels that are activated by ZP3. 3) The role of ZP3-dependent ion channels in the induction of the acrosome reaction will be defined. Selective antagonists will be used to determine the relationship between ZP3-dependent ion channels and internal Ca,+ elevations. 4) The expression during spermatogenesis of ion channels that determine resting membrane potential or that are activated by ZP3 will determined. Health problems are posed when fertilization is either unbounded or is impeded. In this regard, the acrosome reaction is an important regulatory event during mammalian gamete interaction, with unregulated exocytosis associated a failure of fertilization. These studies define sperm ion channels that are essential components of the ZP3 signaling pathway and may provide new targets for the control of fertilization. Highly-resolved profiles of these channels are provided by state-of-the-art fluorescent and electrophysiological methods and may permit rationale design of new antagonists. In addition, identification of spermatogenic stages that express ZP3-regulated channels sets the stage for the molecular cloning and subsequent analysis of channel cDNAs.